Non Technical Summary Tannins are thought to be important chemical defenses in the leaves of trees, as well as many crop plants. However, our recent work has shown that different types of tannins vary in their chemical activities in caterpillars. It may be important for improving plant resistance to know whether only certain types of tannins defend plants against insect pests. The purpose of this study is to determine whether specific tannins in tree leaves are effective defenses against caterpillars and grasshoppers. This work will provide a basis for predicting the effectiveness of tannin-based plant resistance to a range of pest species, providing new tools for breeding and genetic engineering efforts to improve plant resistance.

Goals / Objectives Ellagitannins, which have been largely ignored in the ecological literature, possess unusually high oxidative activities, while high molecular weight gallotannins and condensed tannins have low activities. The proposed research will examine the patterns of production of three major classes of tannins in tree leaves and their biological activities in insect herbivores. This project will compare eight species of deciduous North American trees with the aim of demonstrating the association between tannin composition and the oxidative activities of these tree leaves for caterpillars.

Project Methods EPR spectrometry will be used to measure the oxidation products of phenolics (semiquinone free radicals) to test whether different types of tannins differ in their oxidative activities in caterpillars. Using purified ellagitannins, gallotannins and condensed tannins, we will examine several biochemical and physiological mechanisms potentially linking their oxidative activities and insect performance. A final major project will involve extending this work to grasshoppers, which have a slightly acidic gut pH.

Progress 12/01/09 to 11/30/10

OutputsOUTPUTS: Tannins and low molecular weight phenolic compounds are produced widely by plants as chemical defenses. When they are eaten by many insects, they can oxidize, producing free radicals and quinones, which are potentially damaging to nutrients in the gut. We tested the hypothesis that the oxidation of ingested phenolic compounds inside the midguts of gypsy moth caterpillars causes a decrease in protein nutritional quality. In tests with peroxidase-overexpressing poplar (oxidatively active) compared with untransformed poplar, and in comparisons between sugar maple (oxidatively active) and red oak (oxidatively inactive), we found little evidence that essential amino acids were being damaged by high levels of phenolic oxidation in the gut. Therefore, the detrimental effects of increased phenolic oxidation on caterpillars may be caused by toxic effects instead. Protein is regarded as the most limiting nutrient for insect herbivores. We tested the hypothesis that protein quality is an important factor that can limit insect performance on different trees. We analyzed the amino acid composition of red oak (a good host plant) and sugar maple (a poor host plant) in both the spring and summer. Our findings indicate that feeding in the spring benefits insects, such as the gypsy moth, by providing large quantities of protein. However, the protein quality (essential amino acid composition) of leaves did not differ significantly between the spring and summer, nor did it differ between oak and maple. There are several factors that were measured that would make the lush spring leaves nutritionally superior to mature foliage, but protein quality does not appear to be one of them. We tested the hypothesis that insects feeding in the spring may avoid the chemical defenses (especially tannins) produced by many trees. Contrary to expectation, gypsy moth larvae that fed on lush maple leaves in May were affected by the toxicity of maple leaves as much as those that fed on mature leaves. By comparison, one of their preferred host plants, red oak, produces minimal oxidative stress in larvae in either the spring or the summer. A pathological condition, oxidative stress, is a key process to understand the effects of oxidative plant defenses against pests, such as gypsy moth. We also discovered that protein utilization efficiency drops in half from the spring to the summer. Future work will examine why this occurs, and whether the mechanism(s) could be used to improve plant defenses against leaf-feeding insects. PARTICIPANTS: This work was done by the collaboration of an American insect physiologist (Raymond Barbehenn), a Finnish tannin chemist (Juha-Pekka Salminen), and a Canadian plant molecular biologist (Peter Constabel). There were five undergraduate students who were trained in biological research, and who will be, or are now, co-authors on research publications. In addition, the work involved the expertise of a graduate student, a postdoctoral fellow, and a senior technician. TARGET AUDIENCES: The results of our work are targeted to other researchers in the fields of plant genetics, ecology, entomology, and plant chemistry. Results from our research were presented at several scientific meetings, as well as at invited talks at several universities in the US, Canada, and Finland. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

ImpactsThe aim of our work is to find new mechanisms for improving plant resistance to pest insects. The strategy involves understanding some of the successful defenses that plants have already evolved, and using these to our benefit in agriculture and forestry. The results of this study point to the importance of improving oxidative defenses, such as certain tannins, in plants. Our work also shows that there may be ways to limit protein digstibility, such as occurs when leaves become mature. Could this process be increased to make leaves even less digestible to insect pests

OutputsOUTPUTS: We examined whether tannins and oxidative enzymes can function together synergistically to help increase plant defenses against caterpillars. It was found that two of the major types of tannins in plants (ellagitannins and condensed tannins) can be oxidized by the plant enzyme horseradish peroxidase (POD). Therefore, the large amounts of tannins in many tree leaves, and not just the smaller amounts of low molecular weight phenolics, are substrates. Tannin oxidation by POD produces free radicals and quinones, which are potentially damaging to nutrients in the gut and to insect tissues. Ellagitannins were successfully oxidized by POD in gypsy moth caterpillar guts, and higher levels of tannin oxidation by POD were associated with decreased caterpillar growth. These results suggests that ellagitannins and POD can function together to improve plant defenses. However, the activity of POD is negligible in the gut unless the tree leaves have been fed upon for a day to induce their production of hydrogen peroxide, which is needed by POD to oxidize the tannins. We are currently testing the hypothesis that tannin oxidation in the gut damages essential amino acids, which could lead to poor insect nutrition. We previously discoverd that gypsy moth larvae that feed on sugar maple leaves have an extremely poor ability to utilize its leaf protein. We tested the hypothesis that this is related to a boost in uric acid (waste nitrogen) production by larvae on maple. Chemical analyses of uric acid produced by larvae on either maple or red oak showed that uric acid levels were actually higher on oak than on maple, opposite of what was expected. Future work will continue to understand how maple prevents its protein from being utilized well, since this mechanism could be a way to improve the resistance of other plants to insect pests. These results will be submitted for publication in January 2010, and a poster will be presented at two national meetings. PARTICIPANTS: PI participation: Raymond Barbehenn, Juha-Pekka Salminen, C. Peter Constabel Technicians: Lynn Yip Graduate students: Lan Tran Undergraduate students: Chris Dukatz, Chris Holt, Austin Reese Organizations involved: University of Michigan, University of Turku (Finland), University of Victoria (British Columbia) Undergraduates and graduate students did such extensive work on the project that they are co-authors on a major paper. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

ImpactsThe aim of our work is to find new mechanisms for improving plant resistance to pest insects. The results of this study show that ellagitannins are more effective defenses when they are present along with high levels of POD in tree leaves. An understanding of the ability of sugar maple to limit protein utilization may be useful for developing new traits for plant breeding or genetic engineering to improve resistance.

Publications

No publications reported this period

Progress 12/01/07 to 11/30/08

OutputsOUTPUTS: In a comparison of the resistance of red oak and sugar maple to gypsy moth caterpillars, we discovered that (1) sugar maple leaves produce significantly higher levels of oxidative stress in the midgut and (2) protein digestibility in sugar maple leaves is among the lowest ever found. Since the addition of similar amounts of ellagitannins to oak as are found in sugar maple did not significantly improve the resistance of oak, it appears likely that a combination of oxidative and nutritional stress from maple is needed to produce an effective defense against gypsy moth caterpillars. It remains to be determined how maple limits the digestibility of its protein. In a study that examined the dose-response effects of hydrolyzable tannins on poplar leaves (tannin-free), we found that over 7.5% (dry weight) tannin was needed to produce a build-up of potentially harmful free radicals from tannin oxidation in the midgut of gypsy moth caterpillars. Such large amounts of tannin are commonly found in tree leaves, and our results suggest that a high dose may be necessary to increase oxidative stress in caterpillars. However, it was also found that caterpillars could overcome the potential negative effects of large amounts of tannins by increasing their consumption rates on tannin-coated leaves. PARTICIPANTS: This work was done by the collaboration of an American insect physiologist (Barbehenn) and a Finnish tannin chemist (Salminen). The two PIs enlisted the full-time help of five undergraduates, three of whom were recognized as co-authors on two papers. In addition, one masters student and two PhD students were also supported by the grant. Each student was trained to a high level in at least one method that was critical to the success of the project. TARGET AUDIENCES: The research papers produced by this work are aimed primarily at other researchers in the fields of plant ecology, agriculture, insect ecology, and tannin chemistry. In addition to producing high-level publications, the results of the work were disseminated at the annual meeting of the Entomological Society of America. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

ImpactsThis work is aimed at informing plant breeders about the most effective types and levels of tannins to increase resistance to pest insects in trees and tannin-producing crops. Our results show that, although ellagitannins are the best type of tannins for producing oxidative stress in caterpillars, ellagitannins alone are unlikely to provide adequate resistance in trees against pest species that are well-adapted to tannins, such as gypsy moths. We hypothesize that a synergistic strategy, whereby both oxidative stress and nutritional stress are exacerbated, may be effective.

Outputs OUTPUTS: There were three major accomplishments during the first year of this project: (1) the tannin (chemical) compositions of the leaves of eight deciduous tree species were analyzed, allowing for a comparison of the amounts of oxidation in the guts of white-marked tussock moth caterpillars, (2) gram quantities of tannins were purified from tree leaves for use in experiments and (3) ellagitannins and condensed tannins were tested as defenses on tree leaves against gypsy moth caterpillars. Tree species with high levels of ellagitannins, such as maples, were found to produce higher levels of potentially damaging oxidation in the tussock moth caterpillars that ate these species of tree leaves. When hybrid poplar leaves were treated with ellagitannins to increase their resistance, the levels of oxidation in the midguts of gypsy moth caterpillars were substantially increased, consistent with a negative impact on the caterpillars. However, these caterpillars were able to compensate for
higher levels of oxidation in their guts by eating more food, thereby avoiding any measurable impact on their growth rates. Similar results were obtained when red oak leaves were coated with ellagitannins. In a third set of experiments, the addition of condensed tannins to sugar maple leaves showed that this type of tannin is much less effective at producing oxidative damage in the gut. Not surprisingly, no impact of condensed tannins on gypsy moth caterpillars was found. We conclude that although ellagitannins are the best type of tannins for producing oxidation in caterpillars, ellagitannins alone are unlikely to provide adequate resistance in trees against pest species that are well-adapted to tannins, such as gypsy moths. Three undergraduate students and one masters student were trained to do the research reported here.
PARTICIPANTS: At the University of Michigan, three undergraduate students and one masters student were trained to do the research reported here. The PI trained the students and led the work on a daily basis. At the co-PI's lab at the University of Turku (Finland), the co-PI and two students worked on chemical aspects of the project.

ImpactsThis work was aimed at informing plant breeders about the most effective types and levels of tannins to produce resistance to pest insects in trees and some crop plants. Our results show that, although ellagitannins are the best type of tannins for producing oxidation in caterpillars, ellagitannins alone are unlikely to provide adequate resistance in trees against pest species that are well-adapted to tannins, such as gypsy moths. Further work will examine whether ellagitannins are successful resistance factors against other, less tolerant pest species.